Project Summary In the bone marrow, hematopoiesis is dependent upon support from the surrounding microenvironment, or niche. The bone marrow microenvironment is complex, with hematopoietic and mesenchymal cell populations interacting to influence the formation of hematopoietic cells, blood vessels and bone. In order to understand the molecular and cellular mechanisms by which multiple cell populations support hematopoiesis, it would be helpful to analyze a large number of cell types simultaneously. Mass cytometry (CyTOF) is a novel technique in which flow cytometry is performed using antibodies coupled to rare earth metal isotopes rather than fluorochromes, followed by mass spectrometry. Unimpeded by spectral overlap, CyTOF allows for the analysis of >40 simultaneous parameters. Using CyTOF we have recently published reference maps of the major hematopoietic cell populations in mouse and human bone marrow. We now propose to expand these to include the mesenchymal populations. Our central hypothesis is that alterations in the distribution and function of mesenchymal populations reciprocally influence bone marrow hematopoiesis. As a corollary, we suggest that bone pathologies affecting mesenchymal cells, such as osteoporosis, will necessarily perturb hematopoietic cell development and function. The parathyroid hormone (PTH) receptor (PTH1R) is a G protein coupled receptor whose signaling in bone has profound effects on bone formation and hematopoiesis. To investigate how PTH signaling alters the bone marrow microenvironment we propose the following aims: In Aim 1 we will expand the reference map of hematopoietic cells in murine bone marrow to include mesenchymal populations by incorporating antibodies to identify endothelial cells, mesenchymal stem/progenitor cells, and osteoblasts. We will further identify major cell type-specific cytokines and signaling pathways stimulated by PTH. In Aim 2 we will examine how bone marrow populations are altered by disruption of PTH/PTH1R/Gs? signaling. We have demonstrated that mice lacking the Gs? subunit, which mediates many PTH-dependent actions in bone, in osteoblast progenitors (Gs?-OsxKO mice) exhibit severe osteoporosis, loss of B lymphocyte precursors, and a failure to increase bone mass in response to PTH. By comparing the frequencies of hematopoietic and mesenchymal populations in Gs?-OsxKO bone marrow to the reference map generated in Aim 1, we will reveal how the absence of Gs? signaling in osteoblasts impacts the bone marrow ecosystem. We will also examine the alterations in cytokine production and PTH-dependent signaling within the bone marrow of Gs?-OsxKO mice. In Aim 3 we will generate a reference map of the human bone marrow microenvironment. Understanding the structure of normal mesenchymal populations in human bone marrow is foundational to examining alterations in disease, and such a reference map will be of value in studying the role of bone marrow microenvironment in human disease, aging, and in response to bone- and blood-targeting medications.